This invention broadly relates to an immunological reagent solution which is useful for enhancing the extent of an immunoprecipitation reaction. More specifically, the invention relates to a reagent solution which can be utilized in a wide variety of immunological assaying methods which involve the reaction of an antigen and antibody to form an antigen-antibody complex, to substantially increase the extent of insolubilization of the antigen-antibody complex.
As generally appreciated by those skilled in the art, there are numerous immunological assaying methods of widely varying methodology which necessitate at one stage or another in the assay, and for varying purposes, the insolubilization of as much of the antigen-antibody complex as possible. For example, complex insolubilization plays an important role in such illustrative important conventional immunological assaying techniques as electrophoretic analysis, enzymatic assays, radioimmunoassays (RIA) and nephelometric assays, and much work has been directed toward developing means for increasing complex insolubilization in order to improve these assays. Normally, complex insolubilization is necessary in order to isolate the immunological reaction product from the unreacted immunological reactants involved in a particular assay so that either the isolated reaction product or the unreacted reactants can be separately analyzed to provide a meaningful diagnostic assay.
While the invention can be utilized in connection with a wide variety of immunological assaying methods, it is particularly useful in carrying out nephelometric analyses. Nephelometric, or light scattering, principles have been adapted to the determination of relatively small quantities of biological constituents which are made to exist in the form of suspended particles such as, for example, by the formation of an antigen-antibody complex. According to this method, a light source is made to pass through a liquid test sample whereby the light rays are directed through the suspended particles of the formed complex. As these light rays strike the particles, they are scattered or diffused at any predetermined angle, for example, at a right angle, from the axis of the bean and are received by photocells. This scattered light is converted to an electrical signal which is directly proportional to the amount of particulate concentration which, in turn, is thereby accurately measured on a meter face of an instrument.
The general principles of naphelometric immunological assays are well known. Examples of instruments suitable for nephelometric analyses are the Hyland Laser Nephelometer PDQ.TM. (Hyland Laboratories); the Aminco-Fluorocolorimeter (American Instrument Company); the Aminco-Bowman Spectrophotofluorometer (SPF); and the Auto Analyzer II with attached Fluoronephelometer (Technicon Instruments Corporation).
By means of these nephelometric principles and equipment, the clinical technologist can make an accurate determination of small concentrations of a wide variety of specific proteins, for example, the immunoglobulins IgG, IgA, IgM, transferrin, complement C3, haptoglobin, alpha.sub.1 -antitrypsin, .beta.-lipoprotein, albumin, alpha.sub.2 -macroglobulin, alpha.sub.1 -acid glycoprotein, and various other biological constituents such as triglycerides, lipoproteins, and human chorionic gonadotropins.
Since the test range and sensitivity of a nephelometric analysis in many cases depend in large part on the extent to which the antigen-antibody complex can be insolubilized, procedures have been developed recently for improving a nephelometric analysis in this respect by use of the polymer polyethylene glycol (PEG). See, for example, Hellsing, Protides Biol. Fluids, Proc. Colloq. 21, pp. 579-83 (1973); Lizane and Hellsing, Clin. Chem. 20, pp. 415-20 (1974); Savory et al., Clin. Chem. 20, pp. 1071-75 (1974); and Tiffany et al., Clin. Chem. 20, pp. 1055-61 (1974). According to these procedures, test samples are diluted with a solution of polyethylene glycol polymer prior to incubation of the sample and reading on the nephelometric instrument. The polyethylene glycol improves the nephelometric analysis by increasing the concentration of suspended particles, thereby improving the analysis. In effect, the polyethylene glycol is enhancing the insolubilization of the antigen-antibody complex. Despite this improvement, there remain many biological constituents which cannot be satisfactorily analyzed on a nephelometer because of inadequate test range of sensitivity caused by too low a concentration of suspended particles at the time the light scattering is performed.
It is therefore an object of this invention to provide an improved immunological reagent system which operates to greatly increase the insolubilization of antigen-antibody complexes beyond that obtainable from polyethylene glycol alone, and hence increase the concentration of suspended particles in a nephelometric analysis, and in various other immunological assaying methods which would benefit from improved insolubilization of the antigen-antibody complex.
It is another object of this invention to increase the test range and sensitivity of a nephelometric analysis and of other immunological assaying techniques which would benefit from improved insolubilization of the antigen-antibody complex.
These and other objects of this invention will be apparent to those skilled in the art from a consideration of this specification taken in its entirety.